The present invention relates to a novel preparation for the detection of chlamydial infections using lipopolysaccharides of Re-lipopolysaccharide mutants of gram-negative bacteria.
Chlamydiae are very small, obligately intracellular bacteria which infect birds, man and other mammals. The chlamydiae are classified into two species, Chlamydia trachomatis and Chlamydia psittaci. Chlamydia trachomatis has a high degree of host specificity, being almost completely limited to man; it causes ocular and genitourinary infections of varying severity. In contrast, Chlamydia psittaci strains are rare in man but are found in birds and in wild and domestic animals. C. psittaci is a cause of abortion and epidemics of diarrhea in animals.
While antichlamydial drug therapy exists, many chlamydial infections go untreated because of the limitations connected with the available diagnostic methods. The best known serological methods used for diagnosis of chlamydiae are complement fixation tests, using chlamydial glycolipid (also called a group antigen), counterimmunoelectrophoresis assays and immunofluorescence assays. These methods are described in U.S. Pat. No. 4,118,469, European Patent No. 17,460 and British Patent No. 2,047,889. The isolation of chlamydial antigens and their use as vaccines are described in U.S. Pat. Nos. 4,118,469, 4,039,657, 4,096,035, 4,271,146 and 4,267,170.
A major problem in diagnosing chlamydial infections resides in culturing the chlamydiae. As obligate intracellular bacteria, chlamydiae must be cultured in living cells, e.g. tissue culture or egg preparation, necessitating laboratories which specialize in cell culture. The methods of culturing chlamydiae are characterized by low yields and inevitable contamination by extraneous substances, such as non-chlamydial proteins, from the associated living cells. Although the presence of contaminants is trivial conceptually, such contaminants are important in serologic methods since even trace levels of contaminants can elicit the formation of detectable amounts of antibody. This problem has necessitated the need for control antigens to assess nonspecific reactions in chlamydial serologic methods. The preparation of chlamydial antigen albeit contaminated with extraneous substances is time-consuming, expensive and even possibly risky to the health of the researcher.
If two different antigen molecules happen to have one or more functional groups in common, then these antigens are said to cross-react. Cross-reaction is a tool which has been utilized for classifying groups of closely related bacteria. For example, the 1500 or more varieties of salmonellae have been arranged into serologic groups by cross-reactions, i.e. the antiserum of a strain of a particular group reacts with other strains of that group. The common groups or antigenic determinants responsible for these group-specific reactions are short sequences of particular sugar residues. Cross-reacting groups need not be identical; they only need to be sufficiently similar. For example, antibodies to m-azobenzenesulfonate cross-react with m-azobenzenearsonate.
Rough (R) mutants are a well known class of bacterial mutants (Luderitz et al., Comprehensive Biochemistry, Vol. 26A, pp. 105-228, M. Florkin & E. H. Stotz, Eds., Elsevier, Amsterdam (1968); Makela & Stocker, Annu. Rev. Genetics, Vol. 3, pp. 291-322 (1969); Makela & Stocker in Genetics As a Tool in Microbiology, Glower & Hopwood, Eds., Soc. Gen. Microbiol. Symp. 31, Cambridge University Press (1981)) in which a step in the biosynthesis of LPS is defective, thereby blocking the completion of the molecule. The R mutants are classified into chemotypes according to their LPS structure (FIG. 4 and pages 156-171 in Luderitz et al. 1968, and FIG. 4 in Luderitz et al., Current Topics In Membranes and Transport, Vol. 17, pp. 79-151 (1982). In this classification, a chemotype of Re or a "Remutant" is defined as a mutant whose LPS consists of lipid A and KDO (3-deoxy-D-manno-octulosonic acid, previously called 2-keto-3-deoxy-octonate) with no other monosaccharide substituents. For this reason Re mutants are often referred to as "heptoseless" since they are devoid of even heptose, the monosaccharide that in most LPS is the next unit linked distal to KDO. Such Re mutants were first described in the genus Salmonella (both S. typhimurium and S. minnesota) but have been later found in other species (Escherichia coli and Proteus mirabilis), and can probably be isolated from many others because the LPS structure in the immediate vicinity of lipid A is very conservative throughout the gram-negative bacteria (Luderitz et al. 1982).
It is an object of this invention to detect antibodies to chlamydiae by using Re-lipopolysaccharide (Re-LPS) from Re-mutants of gram-negative bacteria.
Another object of this invention is to detect chlamydiae by using antibodies to Re-LPS from Re-mutants of gram-negative bacteria.
It is also an object of this invention to eliminate the drawbacks and problems described above which are associated with the culturing of chlamydiae.
It is a further object of this invention to eliminate the need for control antigens.